U.S. patent application number 11/920982 was filed with the patent office on 2009-02-12 for light-emitting biomarker.
This patent application is currently assigned to QUIDD. Invention is credited to Marc Massonneau, Pierre-Yves Renard, Anthony Romieu.
Application Number | 20090041669 11/920982 |
Document ID | / |
Family ID | 35840398 |
Filed Date | 2009-02-12 |
United States Patent
Application |
20090041669 |
Kind Code |
A1 |
Renard; Pierre-Yves ; et
al. |
February 12, 2009 |
Light-Emitting Biomarker
Abstract
The invention concerns novel 1,2-dioxetane derivatives of
general formula (I) as defined in the description, capable of
emitting a detectable luminescent signal, their use in a method for
detecting and/or quantizing a physical, chemical or biological, in
particular enzymatic, phenomenon, as well as a kit for implementing
said method.
Inventors: |
Renard; Pierre-Yves; (Paris,
FR) ; Romieu; Anthony; (Rouen, FR) ;
Massonneau; Marc; (Tillieres sur Avre, FR) |
Correspondence
Address: |
OLIFF & BERRIDGE, PLC
P.O. BOX 320850
ALEXANDRIA
VA
22320-4850
US
|
Assignee: |
QUIDD
MONT-SAINT-AIGNAN
FR
UNIVERSITE DE ROUEN
MONT-SAINT-AIGNAN
FR
|
Family ID: |
35840398 |
Appl. No.: |
11/920982 |
Filed: |
May 24, 2006 |
PCT Filed: |
May 24, 2006 |
PCT NO: |
PCT/FR2006/050482 |
371 Date: |
February 15, 2008 |
Current U.S.
Class: |
424/9.6 ; 435/4;
436/172; 549/510 |
Current CPC
Class: |
G01N 33/582 20130101;
C07D 321/00 20130101; A61K 49/0052 20130101; A61K 49/0041 20130101;
A61K 49/0021 20130101 |
Class at
Publication: |
424/9.6 ;
549/510; 436/172; 435/4 |
International
Class: |
A61K 51/04 20060101
A61K051/04; C07D 321/00 20060101 C07D321/00; G01N 21/76 20060101
G01N021/76; C12Q 1/00 20060101 C12Q001/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 27, 2005 |
FR |
05 51396 |
Claims
1-26. (canceled)
27. A compound of general formula (I): S--B-A in which: S
represents a labile structure that can be activated by a physical,
chemical or biological phenomenon; B is a reactive branch, of which
the chemical structure is such that the activation of the labile
structure S induces its intramolecular rearrangement in a suitable
form for releasing a molecule A.sup.-; and A is a chromophore of
formula (IIa): ##STR00015## in which: .diamond. symbolizes the
covalent bond with B; a) R.sub.1, R.sub.2 and R.sub.3 represent,
independently of one another, a hydrogen atom, a hydroxyl radical,
an amino radical, a thio radical, a halogen atom, a
C.sub.1-C.sub.20 hydrocarbon-based chain chosen from an alkyl
radical, an alkoxy radical, a heteroalkyl radical, a cycloalkyl
radical, a heterocycloalkyl radical, an ether radical, an alkenyl
radical, an alkynyl radical, an alkylamino radical, a dialkylamino
radical, an alkylsilyl, dialkylsilyl or trialkylsilyl radical, an
alkylsiloxy, dialkylsiloxy or trialkylsiloxy radical, an alkylthio
radical, a haloalkyl radical, a C.sub.6-C.sub.30 aromatic ring,
chosen from an aryl radical, a heteroaryl radical, an aralkyl
radical, substituted where appropriate; b) R.sub.2 and R.sub.3 may
be together to form an oxo unit; c) R.sub.1 and R.sub.2 may be
linked so as to form a C.sub.4-C.sub.8 ring fused with the
dioxetane ring, where appropriate interrupted by one or more
heteroatoms chosen from O, N and S, and possibly being substituted
by one or more radicals such as defined previously in a) for
R.sub.1, R.sub.2 and R.sub.3, d) R.sub.2 and R.sub.3 or R.sub.1 and
Ar may be linked so as to form a C.sub.4-C.sub.20 spiro ring with
the carbon of the dioxetane entity which bears them, this ring
possibly being monocyclic or polycyclic, saturated or unsaturated,
aromatic, fused or unfused, and where appropriate incorporating one
or more heteroatoms chosen from O, N and S and optionally being
substituted by one or more substituents such as defined previously
in a) for R.sub.1, R.sub.2 and R.sub.3; Ar represents a
C.sub.6-C.sub.30 arylene radical, optionally substituted by one or
more substituents chosen from a hydroxyl radical, a halogen atom,
an oxo unit, an amino radical, an alkylamino radical, a
dialkylamino radical, a thio radical, an alkylsilyl, dialkylsilyl
or trialkylsilyl radical, an alkylsiloxy, dialkylsiloxy or
trialkylsiloxy radical, an alkyl radical, an alkoxy radical, an
alkylthio radical, a carboxy radical, a formyl radical, an alkyl
ester radical, an alkyl ketone radical, a haloalkyl radical, an
aryl radical, an aryl ester radical, an aryl ketone radical, an
arylamino radical, a diarylamino radical; and X represents O, NH or
S; and its derivatives thereof.
28. The compound as claimed in claim 27, wherein R.sub.1 is a
fluorescent radical.
29. The compound as claimed in claim 27, in which B is represented
by a C.sub.1-C.sub.9 alkyl radical, optionally interrupted by a
C.sub.6-C.sub.14 arylene radical and/or by one or more heteroatoms
chosen from O, N, and S, and optionally substituted by one or more
substituents chosen from a hydroxyl radical, an oxo unit, an amino
radical, a halogen atom, an alkyl radical, an alkoxy radical, a
carboxy radical, an alkylamino radical, a dialkylamino radical, an
aryl radical and an aryloxy radical.
30. The compound as claimed in claim 27, in which B is represented
by the formula (III): ##STR00016## in which: .quadrature. and
.diamond. symbolize, respectively, the covalent bond with the
labile structure S and the chromophore A; j is an integer ranging
from 0 to 2; R.sub.4 is a C.sub.1-C.sub.20 hydrocarbon-based chain,
where appropriate interrupted by one or more heteroatoms and/or
space --CO and/or --N(alkyl)-unit(s), placed ortho, meta or para to
the .quadrature.-(CH.sub.2).sub.j-- radical, and chosen from the
--(CH.sub.2).sub.k-.diamond., --(CH.sub.2).sub.k--C(O)--.diamond.,
--CH.sub.2--O--C(O)--N(CH.sub.3)--(CH.sub.2).sub.2--N(CH.sub.3)--C(O)--O
radicals, with k being equal to 1 or 2; R.sub.5 and R.sub.6 are,
independently of one another, a hydrogen atom or a saturated or
unsaturated, linear, branched or cyclic, C.sub.1-C.sub.20
hydrocarbon-based chain chosen from an alkyl radical, an alkoxy
radical, an alkylamino or dialkylamino radical, an alkylthio
radical, or a C.sub.6-C.sub.30 aromatic ring chosen from an aryl
radical, an aryloxy radical, an arylamino radical and an arylthio
radical.
31. The compound as claimed in claim 27, in which Ar represents an
arylene radical of formula (IVa): ##STR00017## in which: .DELTA.
symbolizes the covalent bond with X and * symbolizes the covalent
bond with the dioxetane ring; R.sub.10 represents a hydrogen atom,
an electroactive radical or a fluorescent radical; and R.sub.1 is
chosen from a hydrogen atom or is a C.sub.6-C.sub.10 aryl radical
fused with the phenylene radical.
32. The compound as claimed in claim 27, in which R.sub.1 is a
saturated or unsaturated, linear, branched or cyclic,
C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 alkoxy radical, optionally
comprising one or more atoms chosen from O, N and S, and is
optionally substituted.
33. The compound as claimed in claim 27, in which when R.sub.1 is a
fluorescent radical, it is chosen from phenyl and its derivatives,
naphthalene and its derivatives, anthracene and its derivatives,
pyrene and its derivatives, biphenyl and its derivatives, acridine
and its derivatives, coumarin and its derivatives, xanthene and its
derivatives, phthalocyanine and its derivatives, stilbene and its
derivatives, furan and its derivatives, oxazole and its
derivatives, oxadiazole and its derivatives, nitrobenzoxadiazole
and its derivatives, benzothiazole and its derivatives, fluorescein
and its derivatives, rhodamine and its derivatives, BODIPY and its
derivatives, eosin and its derivatives, erythrosine and its
derivatives, resorufin and its derivatives, quinoline and its
derivatives, carbazole and its derivatives, fluorescent cyanines
and derivatives, carbocyanine and its derivatives, pyridinium salts
and derivatives, a fluorescent complex of lanthanides and its
derivatives, fluorescent proteins and quantum dots.
34. The compound as claimed in claim 31, wherein when R.sub.1
and/or R.sub.10 are a fluorescent radical, they are chosen from
phenyl and its derivatives, naphthalene and its derivatives,
anthracene and its derivatives, pyrene and its derivatives,
biphenyl and its derivatives, acridine and its derivatives,
coumarin and its derivatives, xanthene and its derivatives,
phthalocyanine and its derivatives, stilbene and its derivatives,
furan and its derivatives, oxazole and its derivatives, oxadiazole
and its derivatives, nitrobenzoxadiazole and its derivatives,
benzothiazole and its derivatives, fluorescein and its derivatives,
rhodamine and its derivatives, BODIPY and its derivatives, eosin
and its derivatives, erythrosine and its derivatives, resorufin and
its derivatives, quinoline and its derivatives, carbazole and its
derivatives, fluorescent cyanines and derivatives, carbocyanine and
its derivatives, pyridinium salts and derivatives, a fluorescent
complex of lanthanides and its derivatives, fluorescent proteins
and quantum dots.
35. The compound as claimed in claim 30, in which R.sub.4 is
represented by the --(CH.sub.2).sub.k-.diamond. radical positioned
ortho to the .quadrature.-(CH.sub.2).sub.j-- radical, and j=k=1 and
R.sub.5=R.sub.6=--O--CH.sub.3.
36. The compound as claimed in claim 31, in which R.sub.10 is an
electroactive radical chosen from a halogen atom, a cyano radical,
a nitro radical, a monosubstituted or disubstituted amide radical,
an alkyl radical, an alkoxy radical, a trialkylammonium radical, an
alkylamido radical, an alkylcarbamoyl radical, an ester radical, an
alkylsulfonamido radical, a trifluoromethyl radical, an alkylsilyl,
dialkylsilyl or trialkylsilyl radical, an alkylsiloxy,
dialkylsiloxy or trialkylsiloxy radical, an alkylamidosulfonyl
radical, an alkylsulfonyl radical, an alkyl thioether radical, a
haloalkyl radical, a C.sub.6-C.sub.10 radical chosen from an
aryloxy radical, an arylamido radical, an arylcarbamoyl radical, an
arylsulfonamido radical, an aryl radical, a heteroaryl radical, a
triaryl or alkylarylsilyl radical, a triarylsiloxy radical, an
arylamidosulfonyl radical, an arylsulfonyl radical and an aryl
thioether radical.
37. The compound as claimed in claim 27, in which X represents
O.
38. The compound as claimed in claim 27, wherein the compound
corresponds to the formula (Va): ##STR00018##
39. The compound as claimed in claim 27, of formula (Vb):
##STR00019##
40. The compound as claimed in claim 39, in which
R.sub.10=R.sub.1.dbd.H, R.sub.1=R.sub.5=R.sub.6=--O--CH.sub.3, and
R.sub.2 and R.sub.3 are linked to form a spiro adamantyl radical
with the carbon of the dioxetane ring which bears them, substituted
where appropriate.
41. The compound as claimed in claim 27, in which when S is a
structure that can be activated by a biological entity, S has the
general formula (VI): in which: .quadrature. symbolizes the
covalent bond with B; P represents a substrate that can be
recognized by said entity; and L represents an oxygen, sulfur,
nitrogen, carbonyl or a --CO-Q- functional group, with Q being O, S
or NH.
42. The compound as claimed in claim 41, in which S represents a
structure that is labile by action of an enzyme.
43. The compound as claimed in claim 42, in which when S represents
a structure that is labile by action of a protease, L represents
--CO-Q, with Q being NH, and P represents a substrate recognized by
said protease.
44. The compound as claimed in claim 27, wherein it is a derivative
of formula (VII): D-S--B-A in which: D represents a target
structure.
45. The compound as claimed in claim 44, in which the target
structure D may be chosen from a lectin, an antibody or a fragment
of the latter; a cell receptor ligand; a ligand capable of
interacting with a membrane lipid or phospholipid; a ligand capable
of interacting with a sugar present at the surface of the
cells.
46. A pharmaceutical composition comprising at least an effective
amount of at least one compound as defined in claim 27.
47. A method for detecting and/or quantifying a physical, chemical
or biological phenomenon, using at least one compound as claimed in
claim 27, in which S represents a labile structure that can be
activated by said phenomenon.
48. A method for detecting and/or quantifying a biological entity
comprising at least the steps of: bringing at least an effective
amount of at least one compound as defined in claim 27 in which S
represents a labile structure that can be activated by said
biological entity to be detected and/or quantified, into contact
with a medium assumed to comprise said entity; and measuring the
luminescent signal generated.
49. The method as claimed in claim 48, wherein said compound is:
##STR00020##
50. The method as claimed in claim 48, carried out in vitro
comprising the step of adding a light amplifier chosen from
fluoroscein, bovine albumin, human albumin, polymeric quaternary
onium salts such as poly(vinylbenzyltrimethyl)ammonium chloride
(TMQ), poly(vinylbenzyltributyl)ammonium chloride (TBQ),
poly(vinylbenzyl-dimethyl)ammonium chloride (BDMQ),
poly(vinylbenzyltributyl)phosphonium chloride,
poly(vinylbenzyltributyl)sulfonium chloride,
poly(benzyldimethylvinylbenzyl)ammonium chloride, a sodium salt of
fluorescein, poly(benzyltributyl)ammonium and the sodium salt of
fluorescein.
51. The method as claimed in claim 48, in which said biological
entity to be detected and/or quantified is an enzyme and in that S
in the compound of general formula (I) represents a substrate that
can be activated by said enzyme.
52. A kit for the detection and/or quantification via detection of
a biological entity comprising at least one compound as defined in
claim 27, in which S represents a labile structure that can be
activated by said biological entity to be detected and/or
quantified.
53. The diagnostic method in vivo using a pharmaceutical
composition comprising a compound as defined in claim 27.
Description
[0001] The present invention relates to novel compounds capable of
emitting a detectable luminescent signal, to their use in a method
for detecting and/or quantifying a physical, chemical or
biological, especially enzymatic phenomenon, and also to a kit
enabling the method to be implemented.
[0002] Numerous detection methods used in biochemistry, or in
cellular biology, rely on the generation of a light signal, for
example fluorescence.
[0003] Thus, numerous chromogenic, fluorescent or luminescent tests
are described in vitro for detecting the activities of various
enzymes. Most of these tests rely on the use of donor/acceptor or
donor/quencher pairs for fluorescence resonance energy transfer
(FRET) or the use of pro-fluorophore entities.
[0004] Unfortunately, this method of detection, satisfactory for in
vitro analysis, does not generally prove workable for in vivo
analysis.
[0005] Thus, for in vivo applications, it is necessary to supply an
excitation in the near infrared. The choice of donor/acceptor or
donor/quencher pairs for which the excitations do not overlap thus
becomes extremely more limited (Funovics et al., Anal. Bioanal.
Chem., 2003, 377:956-963).
[0006] In view of being free from this necessity to excite the
fluorescence donor in the near infrared, it has been proposed to
use bioluminescent or chemiluminescent compounds, that is to say
whose luminescence is generated at the end of a chemical or
biological reaction (Laxman et al., Proc. Natl. Acad. Sci. USA,
2002, 99:16551-16555).
[0007] Chemiluminescent compounds, their preparation and their use
have been known for a long time. These so-called "high-energy"
molecules contain sufficient energy to generate carbonyl groups in
an excited electronic state, responsible for the chemiluminescence
phenomenon observed.
[0008] As a representation of chemiluminescent compounds, mention
may be made of compounds derived from dioxetane and more
particularly the derivatives of 1,2-dioxetane. These compounds are
thermally labile and can be decomposed over a wide temperature
range while emitting a light signal. Generally, these compounds
have the following general formula:
##STR00001##
[0009] in which each R substituent corresponds to a
hydrocarbon-based radical optionally comprising one or more
heteroatoms, and two adjacent R groups or one R adjacent to
Ar--X--W and one of the two other R groups or Ar may be linked, X
may represent O, S or NH and W may represent a detachable group,
where appropriate charged.
[0010] Numerous derivatives of 1,2-dioxetane have been synthesized,
among which compounds comprising a spiroadamantyl radical bonded in
position 3 are already used as chemiluminescent substrates. Such
derivatives are especially described in documents WO 96/24849, WO
90/07511 and U.S. Pat. No. 5,330,900. These derivatives of
1,2-dioxetane may comprise a recognition site for an enzyme and may
be activated by an enzyme recognizing this site in order to
generate a luminescent signal.
[0011] More recently, other dioxetane derivatives have also been
developed, described in JP 2002-02038, EP 1 342 724, and US
2004/0077018, in which various radicals were used to stabilize the
dioxetane ring.
[0012] The principle of the chemiluminescence reaction of these
compounds relies on an exchange of electrons (CIEEL, Chemically
Induced Electron Exchange Luminescence) between the phenolate
functional group and the strained dioxetane ring located at the
para position of the phenol aromatic ring (Matsumoto, J. Photochem.
Photobiol. C: Photochem. Rev., 2004, 5:27-53). The generation of an
unstable compound comprising a phenolate leads to rupture of the
strained 1,2-dioxetane unit, stabilized by the R radicals,
following an electron exchange with the phenolate via the CIEEL
phenomenon, resulting in the appearance of an activated ester that
returns to the ground state by emitting a luminescent signal.
[0013] Phenol, by itself, and also its esters with carboxylic
acids, are the most commonly used precursors for generating an
oxygen anion (oxyanion) of phenol in an alkaline aqueous solution.
However, other derivatives of this type have also been synthesized
from 1,2-dioxetane in which the same oxygen atom has been replaced
by a thiol group, an amine group or a carbon (Matsumoto, J.
Photochem. Photobiol. C: Photochem. Rev., 2004, 5:27-53; Sabelle et
al., J Am Chem Soc, 2002, 124(17); 4874-4880; Matsumoto et al.,
Tetrahedron Lett, 2004, 45: 3779-82; WO 96/24849; WO 00/44719).
However, the latter derivatives have a substantially lower quantum
yield and cannot be used in practice.
[0014] As for the present invention, it aims to provide novel
compounds derived from 1,2-dioxetane capable of generating
directly, or by intramolecular fluorescence resonance energy
transfer (FRET), a luminescent signal capable of being detected
through a thickness of layers of biological tissue.
[0015] Another object of the present invention is to provide
compounds derived from 1,2-dioxetane which can be activated by any
chemical, physical or biological phenomenon, and in particular an
enzymatic activity. The invention thus has the advantage of
expressing information resulting from this activation, for example
information of hydrolysis type within the context of an enzymatic
activity, into the appearance of a phenolate or a thiophenate, and
in conferring on the derivatives spectroscopic properties distinct
from those with which they were originally endowed.
[0016] Another object of the present invention is to provide
compounds derived from 1,2-dioxetane capable of being administered
in vivo in order to be able to be used in medical imaging
methods.
[0017] The inventors have thus observed, against all expectations,
that it was possible to combine with a 1,2-dioxetane type backbone,
such as defined previously, a reactive branch that can be activated
by a physical, chemical or biological phenomenon, and especially by
an enzymatic activity, without being detrimental to the emission of
a light signal.
[0018] The reactive branch in question according to the invention
ensures, due to an intramolecular rearrangement, the transfer of
electrons necessary for the activation of the light signal.
[0019] Thus, according to one of its first subjects, the present
invention relates to a compound of general formula (I):
S--B-A
[0020] in which [0021] S represents a labile structure that can be
activated by a physical, chemical or biological phenomenon, [0022]
B is a reactive branch, of which the chemical structure is such
that the activation of the labile structure S induces its
intramolecular rearrangement in a suitable form for releasing a
molecule A.sup.-; [0023] A is a chromophore of general formula
(IIa):
##STR00002##
[0024] in which: [0025] .diamond. symbolizes the covalent bond with
B; [0026] a) R.sub.1, R.sub.2 and R.sub.3 represent, independently
of one another, a radical as defined subsequently, such as for
example a hydrogen atom, an alkyl radical, an alkoxy radical, an
aryl radical, a hydroxyl radical, an oxo unit, an amino radical, a
silyl radical, a halogen atom; [0027] b) R.sub.2 and R.sub.3 may
form an oxo unit; [0028] c) R.sub.1 and R.sub.2 may be linked so as
to form a ring fused with the dioxetane ring, as defined
subsequently; [0029] d) R.sub.2 and R.sub.3 or R.sub.1 and Ar may
be linked so as to form a spiroring with the carbon of the
dioxetane entity which bears them, as defined subsequently; [0030]
Ar represents an arylene radical especially as defined
subsequently; and [0031] X represents O, NH or S, and its
derivatives, such as salts, esters or derivatives obtained by
functionalization of a compound of general formula (I) with at
least one unit intended for targeting.
[0032] According to another subject, the present invention relates
to a compound of general formula (Va):
##STR00003##
[0033] in which: [0034] S represents a labile structure and X, Ar,
R.sub.1, R.sub.2, R.sub.3, R.sub.5 and R.sub.6 are as defined
subsequently.
[0035] According to another of its subjects, the present invention
relates to a compound deriving from the functionalization of a
compound according to the invention with at least one structural
unit favoring, for example, the targeting to a particular cell or
tissue.
[0036] According to yet another of its subjects, the present
invention relates to the use, for the purpose of detecting and/or
quantifying a physical, chemical or biological phenomenon, of at
least one compound according to the present invention, in which S
represents a labile structure that can be activated by said
phenomenon.
[0037] According to yet another subject, the present invention
relates to the use of a compound according to the invention, and
especially of general formula (Va), for preparing a pharmaceutical
composition intended for implementing a diagnostic method in
vivo.
[0038] According to yet another of its subjects, the present
invention relates to a kit for the detection and/or quantification
of a biological entity, especially an enzyme, comprising a compound
of general formula (I), and in particular of general formula (Va),
in which S represents a labile structure that can be activated by
said biological entity to be detected and/or quantified.
[0039] According to yet another of its subjects, the present
invention relates to a method for detecting and/or quantifying a
biological entity, especially an enzyme, comprising at least the
steps of: [0040] bringing at least an effective amount of at least
one compound of general formula (I), and in particular of general
formula (Va), in which S represents a labile structure that can be
activated by said biological entity, especially an enzyme, to be
detected and/or quantified, into contact in a medium assumed to
contain said biological entity; and [0041] measuring the
luminescent signal generated.
[0042] According to yet another of its subjects, the present
invention relates to an in vivo diagnostic method, especially for
medical imaging purposes, comprising the detection and/or
quantification of a biological entity, especially an enzyme, by
means of a compound of general formula (I), and especially of
general formula (Va), in which S represents a labile structure that
can be activated by said biological entity to be detected and/or
quantified.
DEFINITIONS
[0043] In the meaning of the present invention, the expression
"alkyl radical" is understood to mean a saturated or unsaturated,
linear, branched or cyclic, fused or unfused hydrocarbon-based
radical having 1 to 20 carbon atoms, preferably 2 to 12 carbon
atoms and better still 3 to 6 carbon atoms and more particularly 4
carbon atoms, capable of being substituted by radicals as defined
below.
[0044] By way of example, included in this definition are radicals
such as methyl, ethyl, isopropyl, n-butyl, t-butyl, t-butylmethyl,
n-propyl, pentyl, n-hexyl, 2-ethylbutyl, heptyl, octyl, nonyl,
decyl, undecyl, dodecyl, tetradecyl, pentadecyl, hexadecyl,
heptadecyl, octadecyl, nonadecyl or icosanyl radicals.
[0045] In the meaning of the present invention, the expression
"cyclic alkyl radical" is understood to mean an alkylene ring
having 4 to 10 carbon atoms, such as a cyclopropyl, cyclopentyl,
cyclohexyl, cyclohexylmethyl or cycloheptyl ring.
[0046] The expression "polycyclic alkyl radical" is understood to
mean a polycyclic alkylene having 4 to 20 carbon atoms, in
particular 6 to 12 carbon atoms, optionally substituted by 1 to 10
radicals independently chosen from a C.sub.1-C.sub.10, alkyl
radical, especially a C.sub.2-C.sub.6 alkyl radical, a
C.sub.1-C.sub.10 alkoxy radical, especially a C.sub.2-C.sub.6
alkoxy radical, a C.sub.1-C.sub.10 alkylamino radical, especially a
C.sub.2-C.sub.6, alkylamino radical, a C.sub.1-C.sub.10 alkylthio
radical, especially a C.sub.2-C.sub.6 alkylthio radical, a halogen
atom and a C.sub.1-C.sub.10 haloalkyl radical, especially a
C.sub.2-C.sub.6, haloalkyl radical.
[0047] By way of example of a polycyclic radical suitable for
implementing the invention, mention may be made of the adamantyl
radical or the bicyclo[2.2.1]heptyl radical.
[0048] In the meaning of the present invention, the expression
"alkoxy radical" is understood to mean a --OR radical in which the
alkyl residue is a saturated or unsaturated, linear, branched or
cyclic, fused or unfused hydrocarbon-based radical, as defined
above.
[0049] Mention may be made, by way of example, of methoxy, ethoxy,
propoxy, butoxy, n-butoxy, isobutoxy, sec-butoxy, n-pentoxy,
isopentoxy, sec-pentoxy, t-pentoxy, hexyloxy, methoxyethoxy,
methoxypropoxy, ethoxyethoxy, ethoxypropoxy groups and the
like.
[0050] In the meaning of the present invention, the expression
"acyl radical" is understood to mean a saturated or unsaturated,
linear, branched or cyclic, fused or unfused hydrocarbon-based
radical, comprising a C.dbd.O functional group and having from 1 to
20 carbon atoms, in particular from 2 to 12 carbon atoms and
preferably from 3 to 6 carbon atoms and more particularly 4 carbon
atoms, for example a formyl radical, an acetyl radical, a succinyl
radical, a benzoyl radical, a 1-naphthoyl or 2-naphthoyl
radical.
[0051] The hydrocarbon-based chain of the aforementioned radicals
may be interrupted by one or more heteroatoms chosen from O, N and
S, to form, for example, a heteroalkyl radical such as an alkyl
ether radical, an alkyl ester radical or a heterocycle.
[0052] In the meaning of the present invention, the expression
"heterocyclic radical" is understood to mean, for example and
nonlimitingly, a furanyl radical, a thiophenyl radical, a pyrolyl
radical, an oxazolyl radical, an isoxazolyl radical, a thiazolyl
radical, an isothiazolyl radical, an imidazolyl radical, a
pyrazolyl radical, a furazanyl radical, a pyranyl radical, a
pyridinyl radical, a pyridadinyl radical, a pyrimidinyl radical, or
a pyradinyl radical.
[0053] In the meaning of the present invention, the expression
"aryl radical" is understood to mean an aromatic ring system
comprising from 1 to 5 aromatic rings, optionally fused, having 6
to 30 carbon atoms, optionally 6 to 10 carbon atoms, optionally
comprising one or more heteroatoms chosen from O, N and S.
[0054] By way of example of aryl radicals suitable for implementing
the invention, it is possible to mention the phenyl radical,
naphthyl radical, anthryl radical, and all the aromatic rings
comprising one or more heteroatoms chosen from O, N and S, such as
for example pyridine, thiophene, pyrrole, furan, quinoline. In the
meaning of the present invention, the expression "arylene radical"
is understood to mean an aryl radical, as defined previously, which
is bivalent.
[0055] As examples of substituents capable of being used to
substitute the aforementioned radicals, mention may be made of a
hydroxyl radical, an oxo unit, a thio radical, an amino radical, a
halogen atom, an alkyl radical, a carboxy radical, an acyl radical,
an amido radical, an alkoxy radical, an alkylamino or dialkylamino
radical, an alkylthio radical, a haloalkyl radical such as, for
example, a perfluoroalkyl radical, an alkylsilyl, dialkylsilyl or
trialkylsilyl radical, an alkylsiloxy, dialkylsiloxy or
trialkylsiloxy radical, an aryl radical, as defined previously.
[0056] In the meaning of the present invention, the expression
"halogen atom" is understood to mean an atom of F, Cl, Br or I. The
halogen atoms advantageously used in the present invention are
fluorine and chlorine.
[0057] In the meaning of the present invention, the expression
"haloalkyl radical" is understood to mean an alkyl radical as
defined previously, substituted by one or more halogen atoms such
as defined previously, and especially, by way of nonlimiting
example, a perfluoromethyl radical.
[0058] 1,2-Dioxetane Derivatives
[0059] The compounds according to the present invention have the
general formula (I):
S--B-A
and comprise its derivatives, such as salts, esters or
functionalized derivatives with structural elements, especially for
targeting purposes.
[0060] Reactive Branch B
[0061] In the general formula (I) for compounds according to the
invention, B is different from a simple chemical bond and
represents a hydrocarbon-based reactive branch of which the
chemical structure is such that the activation of the labile
structure S by a chemical, physical or biological phenomenon
induces the intramolecular rearrangement of B in a suitable form
for releasing a molecule A.sup.-.
[0062] Advantageously, B is represented by a C.sub.1-C.sub.8,
especially C.sub.2-C.sub.6, and in particular C.sub.3-C.sub.4 alkyl
radical, optionally interrupted by a C.sub.6-C.sub.14, especially
C.sub.6-C.sub.10 arylene radical and/or by one or more heteroatoms
chosen from O, N, and S, and optionally substituted by one or more
substituents as defined previously and especially chosen from a
hydroxyl radical, an oxo unit, an amino radical, a halogen atom, an
alkyl radical, an alkoxy radical, a carboxy radical, an alkylamino
radical, a dialkylamino radical, an aryl radical and an aryloxy
radical. Advantageously, the alkyl radical comprises two carbon
atoms.
[0063] The arylene radical may also be substituted by one or more
substituents as defined previously.
[0064] According to one embodiment variant, the arylene radical is
chosen from a phenylene or naphthalene radical. In the phenylene
radical, the positions of the two bonds of the radical may be in
ortho, meta or para positions. Advantageously, they are in ortho
positions.
[0065] According to one embodiment, the reactive branch B may be
chosen from: [0066] a benzylidene radical; [0067] a
.quadrature.-N(CH.sub.3)--(CH.sub.2).sub.2--N(CH.sub.3)--C(O)-.diamond.
radical, in which .quadrature. and .diamond. respectively symbolize
the covalent bond with the labile structure S and the chromophore
A; or [0068] a derivative represented by the general formula
(III):
##STR00004##
[0069] in which: [0070] .quadrature. and .diamond. symbolize,
respectively, the covalent bond with the labile structure S and the
chromophore A; [0071] j is an integer ranging from 0 to 2, and
advantageously is equal to 1; [0072] R.sub.4 is a C.sub.1-C.sub.20,
especially C.sub.2-C.sub.12 and in particular C.sub.3-C.sub.6
hydrocarbon-based chain, where appropriate interrupted by one or
more heteroatoms and/or --CO and/or --N(alkyl)-unit(s), placed
ortho, meta or para to the .quadrature.-CH.sub.2).sub.j-- radical,
and especially chosen from the --(CH.sub.2).sub.k-.diamond.,
--(CH.sub.2).sub.k--C(O)-.diamond.,
--CH.sub.2--O--C(O)--N(CH.sub.3)--(CH.sub.2).sub.2--N(CH.sub.3)--C(O)-.di-
amond. radicals, with k being equal to 1 or 2, and advantageously
equal to 1; [0073] R.sub.5 and R.sub.6 are, independently of one
another, a hydrogen atom or a saturated or unsaturated, linear,
branched or cyclic, C.sub.1-C.sub.20, especially C.sub.2-C.sub.12
and in particular C.sub.3-C.sub.6 hydrocarbon-based chain chosen
from an alkyl radical, an alkoxy radical, an alkylamino or
dialkylamino radical, an alkylthio radical, or a C.sub.6-C.sub.30,
especially C.sub.6-C.sub.10 aromatic ring chosen from an aryl
radical, an aryloxy radical, an arylamino radical and an arylthio
radical, especially as defined previously.
[0074] According to one particular embodiment, the radicals R.sub.5
and R.sub.6 are, independently of one another, chosen from: [0075]
a hydrogen atom, a C.sub.1-C.sub.6 alkyl radical, a C.sub.2-C.sub.6
alkenyl radical, a C.sub.3-C.sub.6 alkynyl radical, branched or
unbranched, a C.sub.3-C.sub.6 cycloalkyl radical, substituted or
not by one or more substituents as defined previously and
especially chosen from a halogen atom, an alkyl radical, an alkoxy
radical, an aryl radical such as, for example, a benzyloxy radical
or a benzyl radical; and [0076] a C.sub.3-C.sub.8, in particular
C.sub.4-C.sub.7 cyclic, aliphatic, aromatic or heteroaromatic
hydrocarbon-based chain comprising one or more heteroatoms chosen
from O, N and S, optionally substituted by one or more substituents
as defined previously and especially chosen from a halogen atom, an
alkyl radical, an alkoxy radical, and an aryl radical, such as, for
example, a benzyloxy radical, a benzyl radical, a phenyl radical or
a naphthyl radical.
[0077] According to one particular embodiment, when R.sub.4 is
chosen from --(CH.sub.2).sub.k--C(O)-.diamond. and
--CH.sub.2--O--C(O)--N(CH.sub.3)--(CH.sub.2).sub.2--N(CH.sub.3)--C(O)-.di-
amond., then R.sub.4 is in the para position with the
O--(CH.sub.2).sub.j-- radical, j=k=1 and
R.sub.5.dbd.R.sub.6.dbd.H.
[0078] According to another particular embodiment, R.sub.4 may be
represented by --(CH.sub.2).sub.k-.diamond., positioned ortho to
the .quadrature.-CH.sub.2).sub.j-- radical, and j=k=1 and
R.sub.5=R.sub.6=--O--CH.sub.3.
[0079] Advantageously, the reactive branch B may be such as defined
in U.S. Pat. No. 6,271,345, the content of which is incorporated
here by reference.
[0080] According to one particular embodiment, the intramolecular
rearrangement of B leads to the formation of a lactam or a
lactone.
[0081] Chromophore A [0082] In the general formula (I) for the
compounds derived from 1,2-dioxetane according to the invention,
the chromophore A is represented by the general formula (IIa):
##STR00005##
[0083] in which: [0084] .diamond. symbolizes the covalent bond with
B; [0085] a) R.sub.1, R.sub.2 and R.sub.3 represent, independently
of one another, a hydrogen atom, a hydroxyl radical, an amino
radical, a thio radical, a halogen atom, a C.sub.1-C.sub.20,
especially C.sub.2-C.sub.12, and in particular C.sub.3-C.sub.6
hydrocarbon-based chain chosen from an alkoxy radical, an alkyl
radical, a heteroalkyl radical, a cycloalkyl radical, a
heterocycloalkyl radical, an ether radical, an alkenyl radical, an
alkynyl radical, an alkylamino radical, a dialkylamino radical, an
alkylsilyl, dialkylsilyl or trialkylsilyl radical, an alkylsiloxy,
dialkylsiloxy or trialkylsiloxy radical, an alkylthio radical, a
haloalkyl radical, a C.sub.6-C.sub.30, especially C.sub.6-C.sub.10
aromatic ring, chosen from an aryl radical and a heteroaryl
radical, substituted where appropriate; [0086] b) R.sub.2 and
R.sub.3 may be together to form an oxo unit; [0087] c) R.sub.1 and
R.sub.2 may be linked so as to form a C.sub.4-C.sub.8 ring fused
with the dioxetane ring, where appropriate interrupted by one or
more heteroatoms chosen from O, N and S, and possibly being
substituted by one or more radicals such as defined previously in
a) for R.sub.1, R.sub.2 and R.sub.3, [0088] d) R.sub.2 and R.sub.3
or R.sub.1 and Ar may be linked so as to form a C.sub.4-C.sub.20,
especially C.sub.6-C.sub.10 spiro ring with the carbon of the
dioxetane entity which bears them, this ring possibly being
monocyclic or polycyclic, saturated or unsaturated, aromatic, fused
or unfused, and where appropriate incorporating one or more
heteroatoms chosen from O, N and S and optionally being substituted
by one or more radicals such as defined previously in a) for
R.sub.1, R.sub.2 and R.sub.3; [0089] Ar represents a
C.sub.6-C.sub.30, especially C.sub.6-C.sub.10 arylene radical,
optionally substituted by one or more substituents especially
chosen from a hydroxyl radical, a halogen atom, an oxo unit, an
amino radical, an alkylamino radical, a dialkylamino radical, a
thio radical, an alkylsilyl, dialkylsilyl or trialkylsilyl radical,
an alkylsiloxy, dialkylsiloxy or trialkylsiloxy radical, an alkyl
radical, an alkoxy radical, an alkylthio radical, a carboxy
radical, a formyl radical, an alkyl ester radical, an alkyl ketone
radical, a haloalkyl radical, an aryl radical, an aryl ester
radical, an aryl ketone radical, an arylamino radical, a
diarylamino radical; and [0090] X may represent O, NH or S.
Advantageously, X may represent O.
[0091] The compound A.sup.- generated following the activation of
the labile structure S and the intramolecular rearrangement of the
reactive branch B is unstable and reacts while emitting a light
signal by means of a chemiluminescent reaction.
[0092] According to one particular embodiment, R.sub.1 may be a
fluorescent radical or an alkyl radical, as defined previously,
substituted by a fluorescent radical.
[0093] According to another embodiment, Ar may be substituted by a
radical that is fluorescent in itself, or a radical able to confer
a fluorescent character to the whole of the structure or a radical
able to modify the fluorescent properties thereof.
[0094] According to another particular embodiment, Ar and R.sub.1
and/or R.sub.2 and R.sub.3 may be combined to form a cyclic or
polycyclic or spiro-fused alkyl radical which may: [0095] either
comprise at least one double bond or at least one triple bond in
the ring or in the side chain, and optionally one or more
heteroatoms chosen from O, N and S; [0096] or be joined to, or
fused with, a substituted or unsubstituted aromatic ring. When Ar
and R.sub.1 are not bonded together, Ar may be an aryl radical,
such as a phenyl, naphthyl, or anthryl radical, where appropriate
substituted, especially by radicals as defined previously and
R.sub.1 may be as defined previously. Such compounds are described
in greater detail in Application US 2004/0077018, the content of
which is incorporated into the present application by
reference.
[0097] According to another embodiment, R.sub.2 and R.sub.3 are
bonded together to form an adamantyl radical in the spiro position
of the carbon of the dioxetane ring which bears them, where
appropriate substituted, especially by the R.sub.8 and R.sub.9
radicals defined below.
[0098] According to another embodiment, R.sub.1 and R.sub.2 may be
bonded together to form a ring fused with the dioxetane ring, and
comprising, optionally, one or more heteroatoms chosen from O, N
and S. Advantageously, R.sub.1 and R.sub.2 form an oxocyclopentane
fused with the dioxetane. Such compounds are especially described
in Application EP 1 342 724, the content of which is also
incorporated here by reference.
[0099] According to another embodiment, R.sub.1 and R.sub.2 may be
bonded together to form a polycyclic ring system fused with the
dioxetane. Such compounds are described in Application JP
2004-262817, the content of which is incorporated here by
reference.
[0100] According to one particular embodiment variant, the
chromophore A is represented by the general formula (IIb):
##STR00006##
[0101] in which: [0102] .diamond. symbolizes the covalent bond with
B, and [0103] R.sub.1 is as defined previously. Advantageously,
R.sub.1 may be chosen from an alkyl radical, a fluorescent group
and an alkyl radical substituted by a fluorescent group. The alkyl
radical is as defined previously. Advantageously, R.sub.1 may be a
saturated or unsaturated, linear, branched or cyclic,
C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 alkoxy radical optionally
comprising one or more heteroatoms chosen from O, N and S, and
optionally substituted. R.sub.1 may be substituted by radicals as
defined previously. Advantageously, R.sub.1 is a C.sub.1-C.sub.3
alkyl or C.sub.1-C.sub.3 alkoxy radical, preferably a methyl or
methoxy radical. [0104] R.sub.8 and R.sub.9 are, independently of
one another, a hydrogen atom or an electroactive radical; [0105] X
represents O, NH or S. Advantageously X represents 0; and [0106] Ar
represents an arylene radical, as defined previously, or a radical
of general formula (IVa):
##STR00007##
[0107] in which: [0108] .DELTA. symbolizes the covalent bond with X
and * symbolizes the covalent bond with the dioxetane ring. A and *
may be positioned in ortho, meta or para positions. Advantageously,
A and * may be positioned in the meta position; [0109] R.sub.10
represents a hydrogen atom, an electroactive radical or a
fluorescent radical. Advantageously, the electroactive radical may
be capable of modifying the fluorescent properties of the Ar
radical to the form, for example, of an alkylsilyl, dialkylsilyl or
trialkylsilyl radical, an NO.sub.2 radical, an anthryl radical, a
naphthyl radical (for example, see Matsumoto et al., Tetrahedron
Lett, 2002, 43: 8955-58); [0110] R.sub.11 is chosen from a hydrogen
atom or represents an aryl radical, for example a C.sub.6-C.sub.10
aryl radical, fused with a phenylene radical, especially a phenyl
radical to form a naphthylene radical.
[0111] In the meaning of the present invention, the expression
"electroactive group" is understood to mean an electron-acceptor
group or an electron-donor group.
[0112] According to one particular variant, the chromophore A is
such that at least one of the groups R.sub.8 or R.sub.9 or R.sub.10
is an electroactive radical that promotes the solubilization of the
compound of general formula (I) according to the invention in an
aqueous solution.
[0113] As examples of groups that promote the solubilization of the
compound of general formula (I) in an aqueous solution, mention may
be made of ammonium, phosphonium, sulfonium, carboxylic acid,
sulfonic acid, trifluoromethylsulfonyl, methylsulfonyl, cyano and
hydroxy radicals.
[0114] According to another embodiment, R.sub.8 and R.sub.9 are,
independently of one another, an electroactive radical chosen from
a hydroxyl radical, a halogen atom, a cyano radical, an amide
radical, an alkoxy radical, a haloalkyl radical, a phenyl radical,
an alkoxyphenyl radical.
[0115] According to yet another embodiment variant, R.sub.10 is a
hydrogen atom or an electroactive radical advantageously chosen
from a halogen atom, especially a chlorine or fluorine atom, a
cyano radical, a nitro radical, a monosubstituted or disubstituted
amide radical, an alkyl radical, an alkoxy radical, a
trialkylammonium radical, an alkylamido radical, an alkylcarbamoyl
radical, an ester radical, an alkylsulfonamido radical, a
trifluoromethyl radical, an alkylsilyl, dialkylsilyl or
trialkylsilyl radical, an alkylsiloxy, dialkylsiloxy or
trialkylsiloxy radical, an alkylaminosulfonyl radical, an
alkylsulfonyl radical, an alkyl thioether radical, a haloalkyl
radical, a C.sub.6-C.sub.10 radical chosen from an aryloxy radical,
an arylamido radical, an arylcarbamoyl radical, an arylsulfonamido
radical, an aryl radical, a heteroaryl radical, a triarylsilyl or
alkylarylsilyl radical, a triarylsiloxy radical, an
arylamidosulfonyl radical, an arylsulfonyl radical and an aryl
thioether radical.
[0116] According to one particular embodiment, R.sub.8, R.sub.9 and
R.sub.10 are advantageously a hydrogen atom.
[0117] According to another particular embodiment, R.sub.1 or
R.sub.10 may be a fluorescent radical in order to modify, by
intramolecular FRET, the emission wavelength of the luminescent
compound according to the invention.
[0118] For example, these fluorescent radicals may be chosen,
nonlimitingly, from phenyl and its derivatives, naphthalene and its
derivatives such as 5-dimethylaminonaphthalene-1-sulfonic acid and
hydroxynaphthalenes, anthracene and its derivatives such as
9,10-diphenylanthracene and 9-methylanthracene, pyrene and its
derivatives such as N-(1-pyrene)iodoacetamide and hydroxypyrenes,
biphenyl and its derivatives, acridine and its derivatives such as
hydroxyacridines and 9-methylacridine, coumarin and its derivatives
such as 7-dialkylamino-4-methylcoumarin and
4-bromomethyl-7-methoxycoumarin, xanthene and its derivatives,
phthalocyanine and its derivatives, stilbene and its derivatives
such as 6,6'-dibromostilbene and hydroxystilbenes, furan and its
derivatives, oxazole and its derivatives, oxadiazole and its
derivatives, nitrobenzoxadiazole and its derivatives such as
hydroxynitrobenzoxadiazoles, benzothiazole and its derivatives,
fluorescein and its derivatives such as 5-iodoacetamidofluorescein
and fluorescein-5-maleimide, rhodamine and its derivatives such as
tetramethylrhodamine, tetraethylrhodamine and rhodols, BODIPY and
its derivatives, eosin and its derivatives, erythrosine and its
derivatives such as hydroxyerythrosines and
5-iodoacetamidoerythrosine, resorufin and its derivatives such as
hydroxyresorufins, quinoline and its derivatives such as
6-hydroxyquinoline and 6-aminoquinoline, carbazole and its
derivatives such as N-methylcarbazole, fluorescent cyanines and
derivatives such as hydroxycyanines, carbocyanine and its
derivatives such as phenylcarbocyanine, pyridinium salts and
derivatives such as 4-(4-dialkyldiamidostyryl)-N-methylpyridinium
iodate, a fluorescent complex of lanthanides and its derivatives,
fluorescent proteins such as Green Fluorescent Protein (GFP) and
its mutants and quantum dots.
[0119] Advantageously, R.sub.1 and R.sub.10 may advantageously be
chosen from molecules of which the fluorescence emission wavelength
is in the range from 400 to 900 nanometers, especially from 500 to
900, and more particularly from 600 to 900 nanometers, such as for
example rhodamine and its derivatives, BODIPY and its derivatives,
cyanines and derivatives or quantum dots.
[0120] According to another particular embodiment, the compounds
according to the present invention may be of general formula
(Va):
##STR00008##
[0121] in which: [0122] S represents a labile structure, and X, Ar,
R.sub.1, R.sub.2, R.sub.3, R.sub.5 and R.sub.6 are as defined
previously.
[0123] Advantageously, R.sub.2 and R.sub.3 are linked to form a
spiro adamantyl radical with the carbon of the dioxetane ring which
bears them, where appropriate substituted by radicals such as
defined previously, especially by radicals such as R.sub.8 and
R.sub.9.
[0124] Advantageously, R.sub.1, R.sub.5 and R.sub.6 are methoxy
radicals.
[0125] According to another particular embodiment, the compounds
according to the present invention may be of general formula
(Vb):
##STR00009##
[0126] in which: [0127] S represents a labile structure, and Ar,
R.sub.1, R.sub.2, R.sub.3, R.sub.5, R.sub.6, R.sub.10 and R.sub.11
are as defined previously.
[0128] Advantageously, R.sub.2 and R.sub.3 are fused to form a
spiro adamantyl radical position with the carbon of the dioxetane
ring which bears them, where appropriate substituted by radicals
such as defined previously, especially by radicals such as R.sub.8
and R.sub.9.
[0129] Advantageously, R.sub.1, R.sub.5 and R.sub.6 are methoxy
radicals.
[0130] R.sub.10 and R.sub.11 are advantageously a hydrogen atom, or
R.sub.10 is a hydrogen atom and R.sub.1, forms a naphthylene
radical with the phenylene radical.
[0131] According to another particular embodiment, the compounds
according to the present invention may be of general formula
(Vc):
##STR00010##
in which S represents the labile structure and R.sub.1, R.sub.5,
R.sub.6, R.sub.8, R.sub.9, R.sub.10 and R.sub.11 are as defined
previously.
[0132] According to another particular embodiment, the compounds
according to the present invention may be represented by the
general formula (Vc) in which
R.sub.8=R.sub.9=R.sub.10=R.sub.11.dbd.H and
R.sub.11=R.sub.5=R.sub.6=--O--CH.sub.3.
[0133] Labile Structure S
[0134] In the general formula (I) of the compounds according to the
invention, S represents a labile structure that can be activated by
a chemical, physical or biological phenomenon or entity.
[0135] The expression "physical phenomenon" is understood in the
meaning of the present invention to denote, for example, a
temperature variation or a pressure variation.
[0136] The expression "chemical phenomenon" is understood in the
meaning of the present invention to denote a phenomenon involving a
chemical entity modifying the chemical properties of the
surroundings, such as for example a variation of pH via an increase
or decrease in protons, a variation in the salinity via an increase
or decrease in the content of salt(s), a variation in the redox
potential, the appearance or disappearance of chemical species, for
example ions such as F.sup.-, S.sup.-, and alkaline-earth metal
ions such as Ca.sup.2+.
[0137] When S represents a labile structure that can be activated
by a chemical phenomenon, S may advantageously be, for example, an
ammonium, amide, amine, ester, silylane, thiol, carbamate or
carbonate group.
[0138] The expression "biological phenomenon" is understood in the
meaning of the present invention to denote a phenomenon involving
the activity of a biological entity, for example an enzymatic
activity, in the form, for example, of a hydrolysis, transfer of an
amine group, or isomerization.
[0139] Thus, during the activation of the compounds according to
the invention by a biological phenomenon, the compounds are
subjected to the action of a biological entity.
[0140] By way of illustration of a biological entity capable of
activating the compounds according to the invention, mention may be
made, nonlimitingly, of enzymes, ribozymes and abzymes.
[0141] By way of example of enzymes capable of activating the
compounds according to the invention, mention may be made of
transferases, such as aminotransferases; hydrolases such as
esterases (for example alkaline phosphatase), glucosidases,
proteases (for example trypsin, metalloproteinases or caspases);
isomerases.
[0142] When S is a structure that can be activated by a biological
entity, S may advantageously be of general formula (VI):
P-L-.quadrature. [0143] in which: [0144] .quadrature. symbolizes
the covalent bond with B; [0145] P represents a substrate that can
be recognized by said entity; and [0146] L represents an oxygen,
sulfur, nitro unit, carbonyl or a --CO-Q- functional group, with Q
being O, S or NH.
[0147] According to one embodiment, S may represent a structure
that is labile by action of an enzyme, and especially by action of
a hydrolase.
[0148] Advantageously, when S represents a structure that is labile
by action of a protease-type hydrolase, L represents --CO-Q, with Q
being NH, and P represents a substrate recognized by a protease,
such as for example a peptide or pseudopeptide residue.
[0149] By way of example of proteases likely to be suitable for
implementing the present invention, mention may be made,
nonlimitingly, of caspases such as 1a caspase-3, acylaminoacyl
peptidase, aminopeptidase M, penicillin G acylase, thermolysin,
cathepsins B, G, L, metalloproteinases, elastase, subtilisin,
plasminogen activator and urokinase.
[0150] According to one embodiment, when P is a peptide residue, it
comprises at least two amino acid residues, in particular at least
four, in particular at least six and, more particularly at least
eight amino acid residues.
[0151] These amino acids may be natural or synthetic and in L or D
configuration.
[0152] These peptides may be obtained by chemical synthesis or
molecular biology according to the usual methods of a person
skilled in the art.
[0153] By way of example of a peptide residue suitable for
implementing the present invention, mention may be made of the DEVD
tetrapeptide sequence (amino acid written in single-letter code)
which is selectively hydrolyzed from the latter aspartate residue
by the caspase-3.
[0154] According to another embodiment variant, S may represent a
structure that is labile by action of an esterase-type hydrolase,
such as a carboxyesterase, a phosphatase or a lipase.
[0155] In this case, L may represent --CO-Q with Q being O, and P
representing a carbon-based residue (of alkyl chain type so that
the P-L assembly forms a residue of a carboxylic acid, especially
of a fatty acid) recognized by an enzyme of esterase or glucosidase
type, or a phosphate group recognized by a phosphatase-type
enzyme.
[0156] When P is a phosphate group, it may be of formula (VII)
##STR00011##
[0157] in which M.sup.+ represents a cation such as an alkali
metal, such as Na.sup.+ or K.sup.+, an ammonium, or a quaternary
ammonium cation N(R).sub.4.sup.+ in which each R may be a
C.sub.1-C.sub.2 alkyl, an arylalkyl, such as a benzyl, or form part
of a heterocycle, such as a pyridinium ring.
[0158] Such structures are advantageously hydrolyzed by an alkaline
phosphatase.
[0159] According to another embodiment variant, S may represent a
structure that is labile by action of a glucosidase-type
enzyme.
[0160] In this case, L may advantageously be an oxygen atom and P
represents a substrate recognized by a glucosidase, such as a
sugar.
[0161] By way of example of a sugar likely to be suitable for
implementing the invention, mention may be made of .alpha.-D- or
.beta.-D-galactoside, .alpha.-D- or .beta.-D-glucoside, .alpha.-D-
or .beta.-D-mannoside and .alpha.-D- or
.beta.-D-fructofuranoside.
[0162] According to another embodiment, S may represent a structure
that is labile by action of an aminotransferase.
[0163] In this case, L may represent a carbonyl group and P may
represent a substrate recognized by an enzyme such as, for example
and nonlimitingly, an alanine aminotransferase or an aspartate
aminotransferase.
[0164] Targeting
[0165] According to one embodiment, a compound according to the
invention may comprise, in addition, at the labile structure S, a
target structure D.
[0166] Thus, a compound may be of general formula (VII) below:
D-S--B-A
[0167] in which: [0168] D represents a target structure; and [0169]
S, B and A are as defined previously.
[0170] The target structure D, or ligand, is an element that can of
recognize or preferentially bond to cells expressing a particular
cellular element, such as for example, and nonlimitingly, a
receptor, an enzyme, a structural protein, a glycoprotein, a sugar,
a lipid or a phospholipid. Advantageously, the cellular element is
a membrane element, preferably having one part of its structure
positioned in the extracellular medium.
[0171] According to one embodiment, this cellular element may be
specific to a physiological or pathological state of the cell or of
the tissue surrounding the cell or of the organ comprising the
cell. Thus, and nonlimitingly, the cellular element may be specific
to the growth state of the cell, the position in the cell cycle, an
inflammatory response of the cell or of the tissue, apoptosis, or
tissue degeneration.
[0172] The expression "recognize or preferentially bond to" aims to
indicate that the target structure D has a particular affinity for
the cells or tissues in question, even though a nonspecific or less
important bond with other cells or other tissues cannot be
completely excluded in vivo or ex vivo. The preferential bond
however makes-it possible to target the compounds according to the
invention at sites of interest, reducing the dissemination of the
compound according to the invention to tissues and/or cells of less
interest.
[0173] Thus, the target structure D may be chosen, for example and
nonlimitingly, from a lectin, an antibody or a fragment of the
latter that recognizes an element present on the surface of the
cells such as for example a protein, a sugar or a lipid; a cell
receptor ligand such as, for example, a peptide such as the
neuropeptide Y, a catecholamine such as adrenalin, an antagonist
such as, for example, a .beta.-adrenergic receptor antagonist such
as propranolol, a growth factor, an aminoacid or a derivative of
the latter such as, for example, glutamate, a cytokine such as, for
example, an interleukin, an interferon or a TNF, a hormone, a
vitamin, an apoliproprotein or cholesterol; a ligand capable of
interacting with a membrane lipid or phospholipid such as, for
example, an annexin; a ligand capable of interacting with a sugar
present at the surface of the cells.
[0174] The expression "cell receptor" is understood to mean any
cellular element capable of transducing information from the
outside to the inside of the cell.
[0175] It is a matter for the knowledge of the person skilled in
the art to determine the nature of the structure D suitable for the
desired targeting, where appropriate by making use of test methods
commonly practiced in the field.
[0176] According to one embodiment, the target structure D may be a
ligand, for example a polypeptide, capable of bonding to the
surface of cells present in a characteristic or specific way in a
particular tissue or that present a pathology.
[0177] The target structure D may also bond to the surface of tumor
cells or cells present in a tumor tissue, or to the surface of
cells present in an inflammatory tissue.
[0178] The target structure D may, for example, bond to the surface
of cells engaged in an apoptosis process, which expose, on their
surface, negatively charged lipids, such as for example
phosphatidylserine.
[0179] According to one embodiment variant, the target structure D
may be a protein or a protein fragment such as a peptide or a
polypeptide or a pseudopeptide.
[0180] Among the proteins capable of bonding to the membranes
exposing negatively charged lipids, mention may be made,
nonlimitingly, of the annexin family, the families of proteins
comprising a C1 or C2 domain, such as factors V and VIII for blood
coagulation, the families of proteins comprising a PH domain or a
FYVE domain, or else the proteins comprising a domain identical or
homologous to the 5 domain of .beta.2-glycoproteins-I (.beta.GP-I).
These proteins or these domains derived from their sequences may be
used as a target structure in compounds according to the
invention.
[0181] According to another embodiment, one or more compounds of
general formula (I) may be fixed to a target structure D in order
to increase, in particular, the intensity of the signal
emitted.
[0182] Thus, at least one, especially at least two, in particular
at least 3, in particular at least 4, and more particularly at
least 5 compounds of formula S--B-A may be fixed to the target
element D.
[0183] Detection Method
[0184] As specified previously, another subject of the present
invention is the use, for the purpose of detecting and/or
quantifying a chemical, physical or biological phenomenon, as
specified previously, of at least one compound according to the
invention, in which S represents a labile structure that can be
activated by said phenomenon.
[0185] Advantageously, the compound according to the invention
implemented in this use may be of general formula (Va) or (Vb) as
defined previously.
[0186] In particular, the present invention aims to also provide a
method for detecting and/or quantifying a biological entity.
[0187] The method according to the invention comprises at least the
steps of bringing at least an effective amount of at least one
compound according to the present invention, and especially of
general formula (Va) or (Vb) in which S represents a labile
structure that can be activated by said biological entity to be
detected and/or quantified, into contact with a medium assumed to
comprise said entity, and measuring of the luminescent signal
generated.
[0188] The method according to the invention may be carried out in
vitro, ex vivo or in vivo.
[0189] When a medium, in which a chemical, physical or biological
phenomenon is able to occur, is brought together with an effective
amount of at least one compound according to the invention of which
the entity S represents a labile structure that can be activated by
said phenomenon, under suitable conditions for achieving said
phenomenon, the activation of the structure S leads to the emission
of a luminescent signal.
[0190] By detecting the presence or absence of the luminescent
signal, it is possible to demonstrate the presence or absence of
said phenomenon and by measuring the intensity of the signal, the
extent of the phenomenon can also be determined.
[0191] According to one particular embodiment, the biological
phenomenon to be detected/quantified may be activity of a
biological entity, especially an enzyme.
[0192] In this particular implementation, the labile structure S
represents a substrate recognized by said enzyme. The action of the
enzyme on the substrate S, especially on the group L of the general
formula (II), under conditions suitable for the reactivity of the
enzyme leads to the emission of a luminescent signal.
[0193] Thus, within the context, for example, of a hydrolase, the
hydrolysis of an enzymatically hydrolyzable group, especially
represented by L in the general formula (II), under suitable
conditions for the reactivity of the enzyme, leads to the emission
of a luminescent signal.
[0194] By detecting the presence or absence of a luminous signal,
it is possible to demonstrate the presence or absence of the enzyme
and by measuring the intensity of the signal, the concentration
and/or the kinetic characteristics may also be determined.
[0195] Advantageously, the medium which may be suitable for
implementing the method according to the present invention may be a
synthetic or natural biological medium in the form of a sample of
biological fluid.
[0196] It may be advantageous to use, in vitro, or where
appropriate ex vivo, together with a compound according to the
invention, at least one light amplifier capable of increasing the
luminescent signal resulting from the degradation of said compound
according to the invention.
[0197] Such compounds are already known.
[0198] By way of example of these amplifiers, mention may be made
of fluorescein, bovine albumin, human albumin, polymeric quaternary
onium salts such as poly(vinylbenzyltrimethyl)ammonium chloride
(TMQ), poly(vinylbenzyltributyl) ammonium chloride (TBQ)
(Sapphire-II.TM.), poly(vinylbenzyldimethyl)ammonium chloride
(BDMQ) (Sapphire-I.TM.), poly(vinylbenzyltributyl)phosphonium
chloride, poly(vinylbenzyltributyl)sulfonium chloride,
poly(benzyldimethylvinylbenzyl)ammonium chloride, a sodium salt of
fluorescein (Emerald.TM.), poly(benzyltributyl)ammonium and the
sodium salt of fluorescein (Emerald II.TM.).
[0199] When the method according to the invention is carried out in
vitro, the detection of the fluorescent signal may be made by any
machine normally used by persons skilled in the art in this
field.
[0200] For example, a sample brought into contact with a compound
according to the C invention may, optionally after a period of
incubation, be subjected to an analytical method comprising, or
not, a step of separating the elements that make up the sample, for
example by chromatography (for example, high performance liquid
chromatography, HPLC) and a step of spectral analysis (for example,
by UV spectroscopy) of the various fractions from the separation
step. The detection and measurement of the spectroscopic signal
specific to a compound according to the invention (for example, the
height and the surface area of a chromatographic peak) may be
correlated to the presence and/or to the intensity/amount of the
phenomenon to be detected, such as for example the presence, amount
and/or kinetic activity of an enzyme.
[0201] The method, and the use, according to the present invention
may also be advantageously carried out in vivo.
[0202] The compounds according to the present invention may be
first administered to a living organism, such as for example an
animal or a human being, then the detection of the fluorescent
signal may be carried out in vivo according to the methods usually
employed in this domain.
[0203] Advantageously, the in vivo implementation of a use
according to the invention makes it possible to establish images of
susceptible tissues or organs in which said phenomenon to be
detected/quantified is capable of occurring.
[0204] According to one particular embodiment, it is possible to
establish images of the tissues or organs capable of expressing a
biological entity, and especially an enzyme, to be detected and/or
quantified.
[0205] Thus, according to one particular embodiment, the method
according to the invention may be implemented in medical imaging
methods.
[0206] According to another embodiment, the method according to the
invention is an in vivo diagnostic method comprising the detection
and/or quantification of a biological entity, especially an enzyme,
by means of a compound of general formula (Va) or (Vb) in which S
represents a labile structure that can be activated by said
biological entity to be detected and/or quantified.
[0207] The method according to the invention may be applied, for
example and non-exhaustively, for medical, experimental, clinical
or preclinical diagnostic purposes in human beings or in animals
such as laboratory animals or animals used in agriculture, such as
rats, mice, guinea pigs, nonhuman primates, or pigs.
[0208] According to one particular embodiment, the biological
entity capable of being detected and/or quantified by a method
according to the invention may be an enzyme, chosen especially and
nonlimitingly from an aminotransferase or a hydrolase, such as an
esterase, a protease or a glucosidase.
[0209] According to another embodiment, when the enzyme to be
detected and/or to be quantified is a protease, it may
advantageously be chosen, nonlimitingly, from caspases,
penicillinases, carboxypeptidases, trypsin, acylaminoacylpeptidase,
aminopeptidases, cathepsins, metalloproteinases, elastase,
subtilisin, thermolysin, plasminogen activator, urokinase and
isomerases.
[0210] Within the context of in vivo implementation, the compounds
according to the invention may be formed so as to be suitable for
oral, or parenteral, especially intravenous, intraarterial,
intracardial, intracerebroventricular, intraperitoneal, or
intratumoral administration, or for pulmonary, nasal, ophthalmic
and optionally rectal, vaginal or topical administration.
[0211] The compounds according to the invention may thus be used in
a formulation suitable for the method of detection to be carried
out and for the chosen administration route.
[0212] For example, the compounds according to the invention may be
prepared in the form of a tablet, a gel capsule or a concentrated
or unconcentrated aqueous solution. This aqueous solution may
advantageously be sterile.
[0213] According to another embodiment, the compounds according to
the invention may be in solid form, for example in powder form, and
may be prepared in advantageously sterile aqueous solution just
before their administration.
[0214] Thus, according to one of its aspects, the present invention
also relates to the use of a compound according to the invention,
especially of general formula (Va) or (Vb) for manufacturing a
pharmaceutical composition intended for implementing an in vivo
diagnostic method, especially a medical imaging method.
[0215] Thus, according to another of its aspects, the present
invention also relates to a pharmaceutical composition comprising
at least an effective amount of at least one compound according to
the present invention, especially of general formula (Va) or
(Vb).
[0216] Advantageously, the use of a compound according to the
invention for preparing a pharmaceutical composition may take place
in the form of a derivative, such as a pharmaceutically acceptable
salt or ester. Thus, the present invention also relates to the
salts and esters of the compounds according to the invention. By
way of example of an ester of a compound according to the
invention, mention may be made of a succinate, a hemisuccinate, a
malate, a tartrate, or a glycolate of a compound according to the
invention. By way of example of a salt of a compound according to
the invention, mention may be made of a sulfate, a phosphate, a
sodium salt, or a calcium salt of a compound according to the
invention.
[0217] The adjustment of the appropriate effective amounts of the
compound or compounds according to the invention to be used in a
method or a use according to the invention, depends on the
phenomenon, or on the entity, to be detected/quantified (chemical,
physical or biological phenomenon) and on the environment in which
the method according to the invention is carried out (in vitro, ex
vivo or in vivo).
[0218] In vivo, the effective amounts may also be adjusted
depending on the size and the weight of the individual to whom the
method according to the invention is applied or with whom the use
according to the invention is carried out, and also depending on
the targeted organ or tissue.
[0219] The adjustments may be carried out by any methods normally
used by a person skilled in the art.
[0220] In the meaning of the present invention, the expression
"effective amount" is understood to mean the amount that is
necessary and sufficient to obtain the desired effect, namely the
detection and/or quantification of a chemical, physical or
biological phenomenon, or of a biological entity.
[0221] According to another particular embodiment, the present
invention relates to a kit for the detection and/or quantification,
by generation of a luminescent signal, of a chemical, physical or
biological phenomenon, said kit comprising at least one compound
according to the present invention in which S represents a labile
structure that can be activated by said physical, chemical or
biological phenomenon to be detected and/or quantified.
[0222] According to one particular embodiment, the present
invention relates to a kit for the detection and/or quantification
of a biological entity, especially an enzyme, comprising a compound
according to the present invention, and especially of general
formula (Va) or (Vb), in which S represents a labile structure that
can be activated by said biological entity to be detected and/or
quantified.
[0223] According to one particular implementation, the kit
according to the present invention may also comprise at least one
light amplifier such as defined previously.
[0224] The present invention will be better understood from the
following examples.
[0225] These examples are given as illustrations of the invention
and must not be interpreted as limiting the scope of the present
invention.
EXPERIMENTAL SECTION
[0226] Separation by High Performance Liquid Chromatography
(HPLC)
[0227] Several chromatographic systems were used for the analytical
experiments and the purification steps: [0228] System A: reverse
phase HPLC (C18, HYPERSIL GOLD, 5 is, 4.6.times.150) in a
triethylammonium acetate buffer and acetonitrile (TEAA 100 mm, pH
7.0) [75% TEAA (2 min), then linear gradient from 25 to 100% (30
min) of acetonitrile] at a flowrate of 1.0 m/min. Double UV
detection was carried out at 254 and 285 nm. [0229] System B:
reverse phase HPLC (C18, NUCLEOSIL, 5.mu., 10.times.250) in a
mixture of demineralized water and acetonitrile [75% water (5 min),
then linear gradient from 25 to 55% (15 min) and from 55 to 90% (70
min) of acetonitrile] at a flowrate of 5.0 m/min. uV detection was
carried out at 260 mm. [0230] System C: reverse phase HPLC (C18,
HYPERSIL GOLD, 5.mu., 4.6.times.150) in a triethylammonium acetate
buffer and acetonitrile (TEAA 100 mm, pH 7.0) [90% TEAA (2 min),
then linear gradient from 10 to 90% (40 min) of acetonitrile] at a
flowrate of 1.0 ml/min. Double UV detection was carried out at 254
and 285 mm.
Example 1
Reactive Branch
Synthesis of 4,5-dimethoxy-2-[(phenylacetamino)methyl]phenylacetic
acid (7)
##STR00012##
[0232] Finely pulverized potassium hydroxide (2 g, 36 mmol) was
added to a solution of phenylacetonitrile 13 (2 ml, 17 mmol) in
tert-butyl alcohol (20 ml) with stirring. The reaction mixture was
refluxed for 45 min, then cooled to ambient temperature and poured
into an aqueous solution of sodium chloride (50 ml). The product
was extracted by chloroform (CHCl.sub.3). The organic phase was
dried over MgSO.sub.4 and concentrated under reduced pressure to
give phenylacetamide 12 (2.1 g, 94%) in the form of a white powder.
M.p.=152.degree. C. .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta.
7.40-7.10 (m, 5H), 5.88 and 5.39 (br s, NH.sub.2), 3.50 (s, 2H).
.sup.13C NMR (CDCl.sub.3, 75 MHz) .delta. 173.8, 134.9, 129.5,
129.1, 127.5, 43.4.
[0233] A mixture of phenylacetamide 12 (2 g, 15 mmol) in a solution
of potassium carbonate (4%, 2 ml, 0.6 mmol) and an aqueous solution
of formaldehyde (37%, 2 ml, 20 mmol) was refluxed for 15 min (until
completely dissolved). The mixture was cooled and extracted with
CH.sub.2Cl.sub.2 (2.times.30 ml). The combined organic phases were
dried over MgSO.sub.4 and were concentrated under reduced pressure
to give a crude product (1.35 g), recrystallization in toluene (20
ml) gave N-hydroxymethylphenylacetamide 11 (1.1 g, 44%) in the form
of a white powder. M.p. 78.degree. C. .sup.1H NMR (CDCl.sub.3, 300
MHz) .delta. 7.50-7.20 (m, 5H), 6.48 (br s, NH), 4.69 (t, J=7.0 Hz,
2H), 3.67 (t, J=7 Hz, OH), 3.60 (s, 2H). .sup.13C NMR (CDCl.sub.3,
75 MHz) .delta. 172.9, 134.4, 129.5, 129.0, 127.5, 64.2, 43.6.
[0234] A solution of homovanillic acid 10 (2 g, 11 mmol) and
sulfuric acid (500 ml, 9.4 mmol) in methanol (100 ml) was refluxed
for 3 hours. After cooling, a sodium bicarbonate solution was added
to the reaction mixture. The product was extracted with
CH.sub.2Cl.sub.2 (2.times.50 ml). The combined organic phases were
dried over MgSO.sub.4 and concentrated under reduced pressure to
give methyl 4-hydroxy-3-methoxyphenylacetate 9 (2 g, 94%) in the
form of a colorless oil. .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta.
7.00-6.70 (m, 3H), 5.82 (br s, OH), 3.87 (s, 3H), 3.70 (s, 3H),
3.56 (s, 2H). .sup.13C NMR (CDCl.sub.3, 75 MHz) .delta. 172.6,
146.6, 144.8, 125.7, 122.1, 114.5, 111.8, 55.9, 52.1, 40.8.
[0235] Cesium carbonate (6.6 g, 20.2 mmol) and dimethyl sulfate (2
ml, 21 mmol) were added to a solution of methyl
4-hydroxy-3-methoxyphenylacetate 9 (2 g, 10.2 mmol) in anhydrous
DMF (80 ml). The reaction mixture was heated at 90.degree. C. for 3
hours. After cooling, water (20 ml) was added to the mixture and
the product was extracted with diethyl ether (2.times.50 ml).
[0236] The combined organic phases were washed with an aqueous
solution of NaCl (20 ml), dried over MgSO.sub.4 and concentrated
under reduced pressure. Chromatography on a silica gel using
cyclohexane/ethyl acetate (1/1) as eluent mixture gave methyl
2-(3,4-dimethoxyphenyl)acetate 8 (1.7 g, 80%) in the form of a
colorless oil. .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. 6.80-6.50
(m, 3H), 3.71 (s, 3H), 3.69 (s, 3H), 3.40 (s, 2H). .sup.13C NMR
(CDCl.sub.3, 75 MHz) .delta. 172.2, 148.8, 148.0, 126.4, 121.3,
112.3, 111.1, 55.8, 55.7, 51.9, 40.6.
[0237] A precooled solution of acetic acid (20 ml) and sulfuric
acid (730 .mu.l, 14 mmol) was added to a mixture of
N-hydroxymethylphenylacetamide 11 and methyl
2-(3,4-dimethoxyphenyl)acetate 8 (1.5 g, 7.2 mmol). The suspension
was stirred for 2 hours at 0.degree. C. then at ambient temperature
for 12 hours. The reaction mixture was neutralized with a 5M sodium
hydroxide solution at 0.degree. C. The product was extracted with
ethyl acetate (2.times.30 ml). The combined organic phases were
dried over MgSO.sub.4 and concentrated to give methyl
4,5-dimethoxy-2-[(phenylacetamino)methyl]phenylacetate 6 (2 g, 78%)
in the form of a colorless oil which crystallized. M.p.=118.degree.
C. .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. 7.40-7.15 (m, 5H),
6.68 (s, 1H), 6.59 (s, 1H), 6.24 (br s, NH), 4.28 (d, J=5.6 Hz,
2H), 3.76 (s, 3H), 3.71 (s, 3H), 3.57 (s, 3H), 3.51 (s, 2H), 3.47
(s, 2H). .sup.13C NMR (CDCl.sub.3, 75 MHz) .delta. 172.8, 170.8,
148.4 (2C), 135.0, 129.7 (2C), 129.2, 127.5 (2C), 124.9, 113.8,
113.2, 56.3 (2C), 52.7, 44.2, 41.7, 38.3. IR (CHCl.sub.3) 3017,
1732, 1660, 1520, 1466, 1275. Analysis calculated for
C.sub.20H.sub.23NO.sub.5: C=67.21, H=6.49 and N=3.92. Analysis
measured: C=66.88, H=6.56, N=3.97.
[0238] An aqueous solution of lithium hydroxide (0.5 M, 56 ml, 28
mmol) was added to a solution of methyl
4,5-dimethoxy-2-[(phenylacetamino)methyl]phenylacetate 6 (2 g, 5.6
mmol) in methanol (50 ml). The reaction mixture was stirred at
ambient temperature for 12 hours, then neutralized with an aqueous
solution of HCl (2N) and precipitation of the product was observed.
Filtration gave the
4,5-dimethoxy-2-[(phenylacetamino)methyl]-phenylacetic acid 7 (1.5
g, 78%) in the form of a white powder. M.p.=186.degree. C. .sup.1H
NMR ((CD.sub.3).sub.2SO, 300 MHz) .delta. 8.40 (br s, 1H),
7.30-7.20 (m, 6H), 6.80 (s, 1H), 6.73 (s, 1H), 4.18 (d, J=5.6 Hz,
2H), 3.70 (s, 3H), 3.59 (s, 3H), 3.55 (s, 2H), 3.44 (s, 2H).
.sup.13C NMR ((CD.sub.3).sub.2SO, 75 MHz) .delta. 172.8, 170.0,
147.4, 147.3, 136.6, 129.9, 129.0 (2C), 128.3 (2C), 126.4, 125.2,
114.6, 111.9, 55.6, 55.4, 42.5, 39.6, 37.5. Analysis calculated for
C.sub.19H.sub.21NO.sub.5: C=66.46, H=6.16 and N=4.08. Analysis
measured: C=66.52, H=5.92 and N=4.06.
Example 2
Dioxetane Precursor
Synthesis of
2-[1-(3-hydroxy)phenyl)-1-methoxymethylene]tricyclo[3.3.1.1.sup.3,7]-deca-
ne (3a)
##STR00013##
[0240] tert-Butyldimethylsilyl chloride (2.7 g, 18 mmol) was added
to a solution of methyl 3-hydroxybenzoate 4a (2.3 g, 15 mmol) and
imidazole (2.6 g, 37.5 mmol) in anhydrous DMF (5 ml). The reaction
mixture was stirred at ambient temperature for 14 hours under an
argon atmosphere. The mixture was diluted with water (20 ml) and
the product was extracted with n-pentane (2.times.20 ml). The
combined organic phases were dried over MgSO.sub.4 and concentrated
under reduced pressure. Chromatography on silica gel using
cyclohexane/ethyl acetate (v/v:95/5) as eluent mixture gave the
methyl and 3-(tert-butyldimethylsilyloxy)benzoic acid ester 4b (3.7
g, 93%). .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. 7.42 (d, J=8 Hz,
1H), 7.28 (t, J=2 Hz, 1H), 7.07 (t, J=8 Hz, 1H), 6.81 (dd, J=8 Hz,
2 Hz, 1H), 3.68 (s, 3H), 0.78 (s, 9H), 0.1 (s, 6H). .sup.13C NMR
(CDCl.sub.3, 75 MHz) .delta. 166.9, 155.8, 131.6, 129.4, 124.9,
122.7, 121.1, 52.2, 25.7, 18.2, -4.4.
[0241] LiAlH.sub.4 (800 mg) was slowly added to a suspension of
TiCl.sub.3 (THF).sub.3 (16.5 g, 44.6 mmol) in anhydrous THF (15 ml)
at 0.degree. C. After 10 minutes at 0.degree. C., triethylamine
(3.5 ml) was added and the mixture was refluxed for one hour. A
solution of 2-adamantanone 5 (822 mg, 5.47 mmol) and of the methyl
benzoate ester 4b prepared previously (1.2 g, 444.50 mmol) in
anhydrous THF (10 ml) was added dropwise to the mixture. The
reaction mixture was refluxed for 3 hours. Water was added
carefully to the mixture at 0.degree. C., which was then diluted
with diethyl ether. The organic phase was washed with a saturated
solution of NaCl, then dried over MgSO.sub.4 and concentrated under
reduced pressure. Chromatography on silica gel using
cyclohexane/EtOAc (v/v:99/1) as eluent mixture made it possible to
obtain
2-[1-(3-tert-butyldimethylsilyloxy)phenyl)-1-methoxy-methylene]tricyclo[3-
.3.1.1]decane 3b (796 mg, 66%) in the form of a colorless oil.
.sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. 7.09 (t, 1H), 6.81 (d,
1H), 6.70 (m, 2H), 3.20 (s, 3H), 3.14 (br s, 1H), 2.54 (br s, 1H),
1.87-1.67 (m, 12H), 0.91 (s, 9H), 0.14 (s, 6H). .sup.13C NMR
(CDCl.sub.3, 75 MHz) .delta. 155.5, 143.6, 136.9, 131.2, 129.0,
122.6, 121.2, 119.5, 57.6, 39.3, 39.2, 38.9, 37.4, 32.9, 30.5,
25.8, 18.3, -4.70.
[0242] A solution of n-Bu.sub.4NF in THF (2.1 ml, 2.1 mmol, 1.0M)
was added to a solution of
2-[1-(3-tert-butyldimethylsilyloxy)phenyl)-1-methoxymethylene]tricyclo-[3-
.3.1.1.sup.3,7]decane 3b (740 mg, 1.91 mmol) in dry THF (12 ml).
The reaction mixture was stirred for 15 minutes at ambient
temperature. The mixture was diluted in diethyl ether (30 ml) and
washed with a saturated solution of NaCl. The organic phase was
dried over MgSO.sub.4 and concentrated under reduced pressure to
give, as crude product,
2-[1-(3-hydroxy)phenyl)-1-methoxymethylene]tricyclo[3.3.1.1.sup.3,7]decan-
e 3a in the form of a yellow oil. .sup.1H NMR (CDCl.sub.3, 300 MHz)
.delta. 7.19-6.67 (m, 4H), 3.67 (s, 3H), 3.30 (br s, 1H), 3.1 (br
s, 1H), 2.50 (br s, 2H), 1.87-1.64 (m, 10H).
Example 3
Dioxetane
Synthesis of the Chemiluminescent Derivative According to the
Invention (1)
##STR00014##
[0244]
2-[1-(3-Hydroxy)phenyl)-1-methoxymethylene]tricyclo[3.3.1.1.sup.3,7-
]decane 3a (520 mg), triethylamine (800 .mu.l) and
benzotriazol-1-yloxytris(dimethylamino)-phosphonium
hexafluorophosphate (BOP) (950 mg) were added to a solution of
4,5-dimethoxy-2-[(phenylacetamino)methyl]phenylacetic acid 7 (656
mg, 1.91 mmol) in anhydrous DMF (10 ml). The reaction mixture was
stirred at ambient temperature for 6 hours. The mixture was
concentrated under vacuum. Chromatography on silica gel using
cyclohexane/EtOAc (v/v:40/60) as eluent gave the ether of
3-(adamantan-2-ylidene-methoxymethyl)phenyl enol ester of
[4,5-dimethoxy-2-(phenylacetylaminomethyl)phenyl]-acetic acid 2
(606 mg, 53%) in the form of a white powder. M.p. 95.degree. C.
.sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. 7.21 (t, J=7.9 Hz, 1H),
7.15-7.00 (m, 6H), 6.91 (t, J=2.3 Hz, 1H), 6.84 (ddd, J=7.9 Hz, 2.3
Hz, 0.8 Hz, 1H), 6.80 (s, 1H), 6.77 (s, 1H), 4.44 (d, J=5.3 Hz,
2H), 3.88 (s, 3H), 3.85 (s, 2H), 3.81 (s, 3H), 3.53 (s, 2H), 3.30
(s, 3H), 3.25 (br s, 1H), 2.66 (br s, 1H), 1.90-1.55 (m, 12H).
.sup.13C NMR (CDCl.sub.3, 75 MHz) .delta. 150.6, 148.6, 148.5,
142.6, 137.3, 134.9, 132.9, 129.4, 129.1, 128.9, 127.3, 127.1,
124.1, 122.1, 120.4, 113.5, 112.9, 58.0, 56.1, 56.0, 43.9, 41.4,
39.3, 39.1, 38.3, 37.2, 32.2, 30.3, 28.3. M.S. (Maldi-T of,
positive mode) m/z 634.27 [M+K].sup.+, 618.30 [M+Na].sup.+.
Analysis calculated for C.sub.37H.sub.41NO.sub.6: C=74.60, H=6.94
and N=2.35. Analysis measured: C=74.25, H=7.09 and N=2.41.
[0245] Ozone was sparged through a cooled solution (-78.degree. C.)
of freshly distilled triphenyl phosphite (37 .mu.l, 0.14 mmol) in 9
ml of anhydrous CH.sub.2Cl.sub.2 until the appearance of a slight
blue coloration. The reaction medium was then purged with argon
until the blue coloration disappeared. The ether of enol 2
previously synthesized (21 mg, 0.035 mmol) dissolved in 1 ml of
anhydrous CH.sub.2Cl.sub.2 was added at -78.degree. C. The reaction
mixture obtained was stirred at -30.degree. C. for 1.5 hours and
left to heat up to ambient temperature for 2 hours. After a control
HPLC (system A), the reaction mixture was evaporated to dryness.
The oily residue obtained was purified by semi-preparative HPLC
(system B). The fractions containing the product (retention time
T.sub.R=48-50 min) were partially evaporated then freeze-dried to
give the dioxetane 1 in the form of a white powder (around 1 mg,
yield 5%). HPLC (system C), T.sub.R 23.8 min. UV (recorded during
the HPLC analysis) .lamda..sub.max 235 and 281 nm. MS (ESI,
positive mode) m/z 650.0 (M+Na)+(molecular weight calculated:
627.74 for C.sub.37H.sub.41NO.sub.8).
* * * * *